Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.     

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.     

Turnip yellow mosaic virus as a chemoaddressable bionanoparticle

Viruses and virus-like particles (VLPs) have been demonstrated to be robust scaffolds for the construction of nanomaterials. In order to develop new nanoprobes for time-resolved fluoroimmuno assays as well as to investigate the two-dimensional self-assembly of viruses and VLPs, the icosahedral turnip yellow mosaic virus (TYMV) was investigated as a potential building block in our study. TYMV is an icosahedral plant virus with an average diameter of 28 nm that can be isolated inexpensively in gram quantities from turnips or Chinese cabbage. There are 180 coat protein subunits per TYMV capsid. The conventional N-hydroxysuccinimide-mediated amidation reaction was employed for the chemical modification of the viral capsid. Tryptic digestion with sequential MALDI-TOF MS analysis identified that the amino groups of K32 of the flexible N-terminus made the major contribution for the reactivity of TYMV toward N-hydroxysuccinimide ester (NHS) reagents. The reactivity was also monitored with UV-vis absorbance and fluorescence, which revealed that approximately 60 lysines per particle could be addressed. We hypothesized that the flexible A chain contains the reactive lysine because the crystal structure of TYMV has shown that chain A is much more flexible compared to B and C, especially at the N-terminal region where the Lys-32 located. In addition, about 90 to 120 carboxyl groups, located in the most exposed sequence, could be modified with amines catalyzed with 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride (EDC) and sulfo-NHS. TYMV was stable to a wide range of reaction conditions and maintained its integrity after the chemical conjugations. Therefore, it can potentially be employed as a reactive scaffold for the display of a variety of materials for applications in many areas of nanoscience.     

The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.     

Refined structure of southern bean mosaic virus at 2.9 A resolution

The T = 3 capsid of southern bean mosaic virus is analyzed in detail. The beta-sheets of the beta-barrel folding motif that form the subunits show a high degree of twist, generated by several beta-bulges. Only 34 water molecules were identified in association with the three quasi-equivalent subunits, most of them on the external viral surface. Subunit contacts related by quasi-3-fold axes are similar, are dominated by polar interactions and have almost identical calcium binding sites. There is no metal ion on the quasi-3-fold axis, as previously reported. Subunits related by quasi-2-fold and icosahedral 2-fold axes have different contacts but nevertheless display almost identical interactions between the antiparallel helices alpha A. A dipole-dipole type interaction between these helices may produce an energetically stable hinge that allows two types of dimers in a T = 3 assembly. The temperature factor distribution, the hydrogen-bonding pattern, and the contacts across the icosahedral 2-fold axes suggest that one of the dimer types is present in the intact virion and probably also in solution; the other is produced only during capsid assembly. Interactions along the 5-fold axes are mainly polar and possibly form an ion channel. The beta-sheet structures of the three subunits can be superimposed with considerable precision. Significant relative distortions between quasi-equivalent subunits occur mainly in helices and loops. The two dimeric forms and the subunit distortions are the consequence of the non-equivalent subunit environments in the capsid.     

Atomic force microscopy analysis of icosahedral virus RNA

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.     

Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 A

Human rhinovirus 14 has a pseudo T = 3 icosahedral structure in which 60 copies of the three larger capsid proteins VP1, VP2 and VP3 are arranged in an icosahedral surface lattice, reminiscent of T = 3 viruses such as tomato bushy stunt virus and southern bean mosaic virus. The overall secondary and tertiary structures of VP1, VP2 and VP3 are very similar. The structure of human rhinovirus 14, which was refined at a resolution of 3.0 A [R = 0.16 for reflections with F greater than 3 sigma(F)], is here analyzed in detail. Quantitative analysis of the surface areas of contact (proportional to hydrophobic free energy of association) supports the previously assigned arrangement within the promoter, in which interactions between VP1 and VP3 predominate. Major contacts among VP1, VP2 and VP3 are between the beta-barrel moieties. VP4 is associated with the capsid interior by a distributed network of contacts with VP1, VP2 and VP3 within a promoter. As the virion assembly proceeds, the solvent-accessible surface area becomes increasingly hydrophilic in character. A mixed parallel and antiparallel seven-stranded sheet is composed of the beta C, beta H, beta E and beta F strands of VP3 in one pentamer and beta A1 and beta A2 of VP2 and the VP1 amino terminus in another pentamer. This association plays an essential role in holding pentamers together in the mature virion as this contact region includes more than half of the total short non-bonded contacts between pentamers. Contacts between protomers within pentamers are more extensive than the contacts between pentamers, accounting in part for the stability of pentamers. The previously identified immunogenic regions are correlated with high solvent accessibility, accessibility to large probes and also high thermal parameters. Surface residues in the canyon, the putative cellular receptor recognition site, have lower thermal parameters than other portions of the human rhinovirus 14 surface. Many of the water molecules in the ordered solvent model are located at subunit interfaces. A number of unusual crevices exist in the protein shell of human rhinovirus 14, including the hydrophobic pocket in VP1 which is the locus of binding for the WIN antiviral agents. These may be required for conformational flexibility during assembly and disassembly. The structures of the beta-barrels of human rhinovirus 14 VP1, VP2 and VP3 are compared with each other and with the southern bean mosaic virus coat protein.     

Crystallographic structure of the T=1 particle of brome mosaic virus

T=1 icosahedral particles of amino terminally truncated brome mosaic virus (BMV) protein were created by treatment of the wild-type T=3 virus with 1M CaCl2 and crystallized from sodium malonate. Diffraction data were collected from frozen crystals to beyond 2.9 A resolution and the structure determined by molecular replacement and phase extension. The particles are composed of pentameric capsomeres from the wild-type virions which have reoriented with respect to the original particle pentameric axes by rotations of 37 degrees , and formed tenuous interactions with one another, principally through conformationally altered C-terminal polypeptides. Otherwise, the pentamers are virtually superimposable upon those of the original T=3 BMV particles. The T=1 particles, in the crystals, are not perfect icosahedra, but deviate slightly from exact symmetry, possibly due to packing interactions. This suggests that the T=1 particles are deformable, which is consistent with the loose arrangement of pentamers and latticework of holes that penetrate the surface. Atomic force microscopy showed that the T=3 to T=1 transition could occur by shedding of hexameric capsomeres and restructuring of remaining pentamers accompanied by direct condensation. Knowledge of the structures of the BMV wild-type and T=1 particles now permit us to propose a tentative model for that process. A comparison of the BMV T=1 particles was made with the reassembled T=1 particles produced from the coat protein of trypsin treated alfalfa mosaic virus (AlMV), another bromovirus. There is little resemblance between the two particles. The BMV particle, with a maximum diameter of 195 A, is made from distinctive pentameric capsomeres with large holes along the 3-fold axis, while the AlMV particle, of approximate maximum diameter 220 A, has subunits closely packed around the 3-fold axis, large holes along the 5-fold axis, and few contacts within pentamers. In both particles crucial linkages are made about icosahedral dyads.     

Crystal structure of tobacco necrosis virus at 2.25 A resolution

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.     

Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.     

Electrostatic interaction between RNA and protein capsid in cowpea chlorotic mottle virus simulated by a coarse-grain RNA model and a Monte Carlo approach

Although many viruses have been crystallized and the protein capsid structures have been determined by x-ray crystallography, the nucleic acids often cannot be resolved. This is especially true for RNA viruses. The lack of information about the conformation of DNA/RNA greatly hinders our understanding of the assembly mechanism of various viruses. Here we combine a coarse-grain model and a Monte Carlo method to simulate the distribution of viral RNA inside the capsid of cowpea chlorotic mottle virus. Our results show that there is very strong interaction between the N-terminal residues of the capsid proteins, which are highly positive charged, and the viral RNA. Without these residues, the binding energy disfavors the binding of RNA by the capsid. The RNA forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in the shape of a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid. These findings are consistent with experimental observations.     

Nature and specificity of the RNA-protein interaction in the case of the tymoviruses.

Dissociation-reassociation experiments performed with turnip yellow mosaic virus in the presence of various RNAs and polynucleotides were used to investigate the degree of specificity and the contribution of the associated RNA moiety to the stability of TYMV. The results emphasize the importance of strategic cytosine residues spread along the RNA chain. Some insight into the contribution of the protein could be gained from comparison of TYMV and eggplant mosaic virus (EMV), a virus similar to TYMV although its top component contains low molecular mass RNA's able to bind various amino acids. Hydrophobic interactions between protein subunits are less important in EMV than in TYMV, and artificial capsids could be obtained from dissociated EMV coat protein. Whether the capsid is or is not the precursor of the virion in tymovirus morphogenesis is discussed.     

The Structure of Bacteriophage φCb5 Reveals a Role of the RNA Genome and Metal Ions in Particle Stability and Assembly

The structure of the Leviviridae bacteriophage φCb5 virus-like particle has been determined at 2.9 Å resolution and the structure of the native bacteriophage φCb5 at 3.6 Å. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the φCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, φCb5 particles are destabilized in solution with elevated salt concentration. The model of the φCb5 capsid provides an explanation of the salt-induced destabilization of φCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions.Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the φCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The φCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.     

Studies of virus structure by laser-Raman spectroscopy. Turnip yellow mosaic virus and capsids.

Laser-Raman spectroscopy of the turnip yellow mosaic virus (TYMV) and its capsid indicate the following features of the structure and assembly of the virion. The secondary structure of coat-protein molecules in TYMV is comprised of 9 +/- 5% alpha-helix, 43 +/- 6% beta-sheet, and 48 +/- 6% irregular conformation and is not altered by the removal of the RNA from the capsid. Introduction of as many as 200 chain scissions per RNA molecule also does not affect the overall secondary structure of the encapsulated RNA, which is 77 +/- 5% in the A-helix form. Tryptophan and cysteine residues of the coat protein appear to be in contact with the solvent, while only one of three tyrosines per coat protein is available for hydrogen bonding of its p-hydroxyl group with H2O molecules. Both cytosine and adenine residues of TYMV RNA are protonated in substantial numbers near pH 4.5, suggesting elevation of their respective pKa values within the virion. The Raman data are consistent with chemical evidence favoring interaction between protonated bases of RNA and amino acid side chains of coat protein in TYMV.     

Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus

Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.     

Structural refinement and analysis of Mengo virus

The structure of Mengo encephalomyelitis virus was refined at 3 A resolution with a final R-factor of 0.221 and a root-mean-square deviation from idealized bond lengths of 0.019 A for 10 A to 3 A data with F greater than or equal to 3 sigma(F). The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement averaging procedure and constrained by the icosahedral symmetry of the virus. The virus consists of 60 protomers each having three major subunits, VP1, VP2 and VP3, along with one smaller internal protein, VP4. The three major subunits form similar eight-stranded beta-barrel structures. Alterations in the original sequence were found at position 45 in VP1 (Arg to Ala) and at position 58 in VP3 (Met to Val). The residues in loops I and II of VP1 (82 to 102), the "FMDV loop" in VP1 (205 to 213), the flexible loop of VP3 in the putative receptor attachment site (175 to 185) as well as the terminal regions 260 to 268 in VP1, 253 to 256 in VP2 and 13 to 15 in VP4 were built or modified in regions of weak density. The variation in temperature factors at the end of the refinement is over a wide range (from 2 to 80 A2), with the disordered outer and inner regions showing high mobility. Four cis proline residues, 105 in VP1, 85 and 152 in VP2 and 59 in VP3, have been identified. The disulfide bridge Cys86 to Cys88 in VP3 has been characterized. One phosphate ion and 233 water positions were included in the refinement. It is suggested that this phosphate is associated with the receptor attachment site. There are two major hydrogen-bonding networks involving solvent atoms; one involving only the subunits of a protomer, and the other connecting the protomers in a pentamer. The distribution of atom types around the icosahedral symmetry axes shows that the 5-fold channel is more hydrophobic than that along the 3-fold axis and that there are more charged residues around the 2-fold axis. The analysis of contacts between the different subunits supports the assignment of the protomeric unit. The five protomers that form the pentameric unit are held together by interactions involving the smaller VP4 protein and the amino termini of VP1 and VP3. The pentamers are associated by means of the amino-terminal region of the VP2 subunits, the beta F strand of the VP3 subunits, the C terminus of the VP4 subunits and the electrostatic helical (alpha A) interactions of VP2 subunits across the icosahedral 2-fold axes. The superposition of the corresponding subunits of Mengo virus, human rhinovirus 14 and southern bean mosaic virus has provided an improved sequence alignment. The largest structural similarity is between the VP3 subunits of Mengo virus and rhinovirus, while the least similarity is between the VP1 subunits. The various specialized insertions in the different subunits can be associated with specific functional requirements.     

Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus (STMV) and the strains of satellite tobacco necrosis virus (STNV), STNV-C, STNV-1, STNV-2, Escherichia phages MS2, ?X174, α3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs.     

Structure of a T = 1 aggregate of alfalfa mosaic virus coat protein seen at 4.5 A resolution

A T = 1 empty aggregate of alfalfa mosaic virus coat protein had been crystallized in a hexagonal unit cell and its orientation was determined with the rotation function. A single heavy-atom derivative has now been prepared and the position of the two Hg atoms per protein subunit were determined using a systematic Patterson search procedure, given the particle orientation. Phases, initially determined by single isomorphous replacement, were refined by six cycles of electron density averaging and solvent leveling to produce a 4.5 A resolution electron density map. The protein coat is confined between 95 and 58 A radius. The subunit boundary could be delineated easily. It has a central cavity reminiscent of the beta-barrel in other spherical plant viruses, but its topology could not be determined unambiguously. The spherical particle has large holes at the 5-fold axes, consistent with previous observations. The subunits have substantial interactions at the 2 and 3-fold axes. The structure of the elongated particles is discussed in relation to these results.